In-vitro electrophysiological experiment method

Abstract

Provided is an in-vitro electrophysiological experiment method. The method comprises: injecting into a target tissue or organ an rAAV expressing a low-abundance fluorescent protein, the genome of the rAAV expressing a low-abundance fluorescent protein containing any one among the following a)-c): a) a promoter sequence, a fluorescent protein gene sequence and a PEST sequence; b) a promoter sequence, a fluorescent protein gene sequence and an ecDHFR DD sequence; and c) a TRE promoter sequence and a fluorescent protein gene sequence, the TRE promoter sequence being located at the upstream of the fluorescent protein gene sequence; and when the fluorescent protein has been expressed for a period of time, performing an in-vitro electrophysiological experiment on the target tissue or organ. The rAAV expresses a low-abundance fluorescent protein, the accumulation of fluorescent proteins in cells is reduced, the cytotoxicity is reduced, and the influence on experimental results is relatively small. In addition, low-abundance fluorescent proteins make cells easier to observe and distinguish from other fluorescent cells in the surrounding area in experiments.

Description

A method for in vitro electrophysiological experiments Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an in vitro electrophysiological experiment method. Background Art

[0002] Recombinant adeno-associated virus (rAAV) vectors are essential tools in current scientific research and one of the most promising gene delivery vectors for gene therapy. By modifying the capsid or genome structure of wild-type rAAV, researchers can generate a variety of rAAVs with diverse tissue and organ targeting, expression of different exogenous genes, and specialized regulatory capabilities. In neuroscience, researchers often use rAAV vectors carrying fluorescent protein genes to target specific cell types. Fluorescent protein labeling allows researchers to observe and manipulate cells in a targeted manner and, combined with further immunohistochemistry, in situ hybridization, and in vitro electrophysiology, achieves goals such as labeling and tracing neural circuits. As exogenous genes, intracellularly expressed fluorescent proteins may consume excessive cellular resources and accumulate within cells, leading to cytotoxicity and compromising research results. For example, in in vitro electrophysiological experiments in neuroscience, neurons infected with rAAV and expressing fluorescent proteins may exhibit excessively high fluorescence intensity and prolonged accumulation. This, on the one hand, hinders the maintenance of normal electrophysiological properties of the cells, and on the other hand, hinders precise manipulation of individual cells. Therefore, optimizing the properties of fluorescent proteins delivered by rAAV, preventing excessive accumulation of fluorescent proteins, and developing an rAAV that can express low-abundance fluorescent proteins will help in conducting electrophysiological experiments, such as facilitating researchers to locate single cells and maintain good cell activity in patch clamp experiments.

[0003] Summary of the Invention

[0004] In order to meet the demand for expressing low-abundance, low-toxicity fluorescent proteins in existing electrophysiological research, the present invention aims to provide a method for in vitro electrophysiological experiments.

[0005] In order to obtain a recombinant adeno-associated virus with low-abundance fluorescent protein expression ability, it is difficult to reduce expression intensity and toxicity by directly transforming fluorescent protein. Therefore, the present invention attempts to control the accumulation of fluorescent protein by reducing transcription level and increasing post-translational regulation. The PEST sequence is the amino acid sequence of the C-terminal end of mouse ornithine decarboxylase, which is characterized by being rich in proline / proline (P), glutamic acid / glutamic acid (E), serine / serine (S) and threonine / threonine (T), which can promote protein degradation through the ubiquitin-proteasome pathway. Escherichia coli dihydrofolate reductase destabilization domain (ecDHFR DD) is a protein unstable region derived from Escherichia coli, which has the effect of promoting protein degradation. By expressing a fusion protein of fluorescent protein-PEST or fluorescent protein-DD, the half-life of fluorescent protein can be reduced and the expression abundance of fluorescent protein can be controlled at a lower level. In addition, the expression of fluorescent proteins can also be controlled by reducing the transcription level. For example, the tetracycline-responsive element (TRE) promoter is an inducible promoter. When the tetracycline transcription activator tTA is not present in the cell, TRE has extremely low promoter activity, but there is a situation where trace amounts of fluorescent proteins are leaked and expressed. This characteristic of the TRE-tTA system can also be used to control the expression of trace amounts of fluorescent proteins, and the effect of labeling cells that are sparse can be achieved. The present invention constructs the fluorescent protein gene sequence and the low transcription level promoter sequence TRE into an rAAV genome, or constructs the fluorescent protein gene sequence and the gene sequence of an element that promotes protein degradation (PEST sequence or ecDHFR DD sequence) into an rAAV genome, thereby achieving the purpose of preparing rAAV that expresses low-abundance fluorescent proteins.

[0006] One embodiment of the present invention is to construct a fluorescent protein gene sequence and a PEST gene sequence or an ecDHFR DD sequence into the same rAAV genome, so that the fluorescent protein and the PEST peptide or the fluorescent protein and the ecDHFR DD peptide can be expressed as a fusion protein. The rAAV genome also contains a promoter sequence and a transcriptional regulatory sequence to ensure the expression of a complete fluorescent protein fusion protein. At the same time, the rAAV genome can be coated with capsids of different serotypes. Since the serotype of the rAAV capsid determines its different tissue and organ tropism, the rAAV can achieve the purpose of expressing low-abundance fluorescent proteins in specific tissues and organs.

[0007] Another embodiment of the present invention is to construct a TRE promoter sequence and a fluorescent protein gene sequence in the rAAV genome, and the fluorescent protein gene sequence is located downstream of the TRE promoter, so that the TRE promoter can control the transcription of the fluorescent protein gene. In the absence of tTA, the TRE promoter has a weak leakage expression, thereby expressing a small amount of fluorescent protein. The rAAV genome also contains a promoter sequence and a transcriptional regulatory sequence to ensure that the fluorescent protein can be expressed completely through the TRE promoter. The rAAV genome can be coated with capsids of different serotypes. Since the serotype of the rAAV capsid determines that it has different tissue and organ tropisms, the rAAV can achieve the purpose of expressing low-abundance fluorescent proteins in specific tissues and organs.

[0008] The specific technical solutions of the present invention are as follows:

[0009] The present invention provides a method for an in vitro electrophysiological experiment, the method comprising the steps of:

[0010] (1) Injecting rAAV expressing a low-abundance fluorescent protein into the target tissue or organ;

[0011] The rAAV genome expressing a low-abundance fluorescent protein contains any one of the following a)-c): a) a promoter sequence, a fluorescent protein gene sequence, and a PEST sequence; b) a promoter sequence, a fluorescent protein gene sequence, and an ecDHFR DD sequence; c) a TRE promoter sequence and a fluorescent protein gene sequence, wherein the TRE promoter sequence is located upstream of the fluorescent protein gene sequence;

[0012] (2) After the fluorescent protein is expressed for a period of time, an in vitro electrophysiological experiment is performed on the target tissue or organ.

[0013] Furthermore, the in vitro electrophysiological experiment is a patch clamp experiment.

[0014] Furthermore, the PEST sequence in a) is located downstream of the fluorescent protein gene sequence and is located in the same expression frame as the fluorescent protein gene sequence, and the promoter sequence is located upstream of the fluorescent protein gene sequence and the PEST sequence;

[0015] b) The ecDHFR DD sequence is located downstream of the fluorescent protein gene sequence and is located in the same expression frame as the fluorescent protein gene sequence, and the promoter sequence is located upstream of the fluorescent protein gene sequence and the ecDHFR DD sequence.

[0016] Furthermore, the PEST sequence is shown as SEQ ID NO.1, the ecDHFR DD sequence is shown as SEQ ID NO.2, and the TRE promoter sequence is shown as SEQ ID NO.1.

[0017] Furthermore, the rAAV genome expressing the low-abundance fluorescent protein also contains a transcriptional regulatory sequence.

[0018] Furthermore, the fluorescent protein is selected from any one of eGFP, BFP, CFP, GFP, YFP, RFP, iRFP, Cerulean, Venus, eGFP, eCFP, eYFP, eBFP, DsRed, dTomato, tdTomato, mCherry, mKate, mApple, mBanana, mCitrine, mOrange, mPlum, tagRFP, tagBFP, and HRP.

[0019] Furthermore, the promoter is selected from any one of CAG, CaMKIIα, CAR, CBA, CD68, c-fos, ChAT, CMV, CR, Ef1α, E-SARE, GAD67, GFAP, GFAP104, gfaABC1D, Grm6, hGRK1, hSyn, hUbC, LP1B, L7 / Pcp2, MBP, MCK, mDlX, mOXT, mTH, nEF, Nestin, NPY, Nrl, PGK, PV, RAM, RK, ROH, RPE65, SFRP2, SST, TBG, TCAP, TH, Thy1, TPH2, TRE, TRPV1, UAS, and Vgat.

[0020] Furthermore, the regulatory sequence is selected from any one of the transcriptional and post-transcriptional regulatory sequences WPRE, oPRE, cw3sl, SV40 polyA, hGH polyA, bGH polyA, and rbGlob polyA.

[0021] Furthermore, the serotype of the rAAV expressing the low-abundance fluorescent protein is selected from any one of AAV1, AAV2, AAV2-Retro, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9-Retro, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV-PHP.eB, AAV-myo and derivative serotypes thereof.

[0022] Further, the tissue is selected from neural tissue, muscle, epithelial tissue or connective tissue;

[0023] The organ is selected from the group consisting of bladder, eye, ear, nose, mouth, tongue, pharynx, larynx, vomeronasal organ, salivary gland, liver, kidney, spleen, heart, intestine, stomach, pancreas, lung, trachea, blood vessel, lymphatic vessel, lymph node, limb, pituitary gland, thyroid gland, parathyroid gland, pancreatic islet, adrenal gland or reproductive organ.

[0024] Furthermore, the neural tissue is selected from the central nervous system and its neural circuit-related tissues.

[0025] The present invention has the following advantages:

[0026] By constructing a fluorescent protein gene sequence and a low transcription level promoter sequence into one rAAV genome, or by constructing a fluorescent protein gene sequence and a gene sequence of an element that promotes protein degradation into one rAAV genome, rAAV expressing low-abundance fluorescent protein can be prepared, solving the problems of excessive expression intensity of fluorescent protein in cells, long accumulation time, cytotoxicity, affecting research results, and inconvenience in precise manipulation of single cells. Specifically:

[0027] 1. The rAAV of the present invention expresses a low-abundance fluorescent protein, reducing intracellular fluorescent protein accumulation and cytotoxicity, with minimal impact on experimental results. This invention does not modify the fluorescent protein itself, but rather combines any existing fluorescent protein with the TRE promoter, PEST, or ecDHFR DD to achieve the goal of reducing the abundance of fluorescent expression. It is generally applicable to fluorescent proteins of all colors.

[0028] 2. The low abundance of fluorescent proteins in the present invention makes it easier to observe and distinguish cells from other fluorescent cells in the surrounding area during experiments. Therefore, the rAAV in the present invention is helpful for in vitro electrophysiological experiments such as patch clamp. For example, in patch clamp experiments on fluorescently labeled neurons, the sparse and moderately bright fluorescence helps researchers accurately locate the cell membrane and record its electrophysiological properties.

[0029] 3. The present invention combines different capsid serotypes, and the rAAV expressing low-abundance fluorescent protein is suitable for a variety of tissues and organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG1 is a schematic diagram of the genome structure of rAAV expressing a low-abundance fluorescent protein.

[0031] Figure 2 shows the fluorescence expression results of HEK-293T cells infected with three adeno-associated viruses expressing low-abundance fluorescent proteins, rAAV-P1, rAAV-P2, and rAAV-P3, and two control viruses, rAAV-C1 and rAAV-C2, after 24, 48, and 72 hours. rAAV-P1 and rAAV-P2 labeled more cells with weaker fluorescence, while rAAV-P3 labeled cells more sparsely and with weaker fluorescence. DETAILED DESCRIPTION

[0032] To more clearly understand the present invention, the present invention is further described with reference to the following examples and accompanying drawings. The examples are intended to illustrate the present invention only and are not intended to limit the present invention in any way. In the examples, all raw materials and reagents are commercially available. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0033] Example 1: Design of rAAV genome and preparation of rAAV

[0034] 1. Construct plasmids carrying the core components of the rAAV genome. The plasmids are:

[0035] P1: pAAV-Ef1α-mCherry-PEST, in which mCherry-PEST is in the same expression frame.

[0036] P2: pAAV-Ef1α-mCherry-ecDHFR DD, in which mCherry-ecDHFR DD is in the same expression cassette.

[0037] P3: pAAV-TRE-mCherry.

[0038] The core plasmid of the present invention was commissioned to Brinkasse (Shenzhen) Biotechnology Co., Ltd. for construction, wherein the PEST sequence (SEQ ID NO.1), ecDHFR DD sequence (SEQ ID NO.2), and TRE sequence (SEQ ID NO.3) are synthetic sequences. After synthesis, the PEST sequence and ecDHFR DD sequence were constructed downstream of mCherry and located in the same expression frame of mCherry; TRE was constructed upstream of mCherry.

[0039] 2. Prepare rAAV using a three-plasmid packaging system. For core plasmids P1, P2, and P3, each core plasmid was co-transfected with the adenovirus helper plasmid pAd-Helper and the AAV capsid plasmid pAAV-RC2 / 9 at the same molecular number into HEK-293T cells. Cell culture was collected 72 hours after transfection, concentrated and purified using iodixanol density gradient centrifugation, and rAAV titers were detected using SYBR Green qPCR. Three types of rAAV were finally obtained. The rAAVs are:

[0040] rAAV-P1: rAAV9-Ef1α-mCherry-PEST, the virus titer is 5.9×10 12 VG / mL;

[0041] rAAV-P2: rAAV9-Ef1α-mCherry-ecDHFR DD, the virus titer is 5.6×1012 VG / mL;

[0042] rAAV-P3: rAAV9-TRE-mCherry, the virus titer is 5.9×10 12 VG / mL.

[0043] SEQ ID NO.1: 120 bp

[0044] SEQ ID NO. 2: 477 bp

[0045] SEQ ID NO.3: 315 bp

[0046] Example 2: rAAV cell infection

[0047] HEK-293T adherent cells were seeded in a 96-well plate at a density of 1 / 3 and allowed to adhere and grow for 24 hours. After 24 hours, the cell culture medium was replaced with a medium containing rAAV-P1, rAAV-P2, and rAAV-P3 viruses. The amount of virus added to the culture medium was sufficient to achieve a multiplicity of infection (MOI) of 1×10 5 , that is, the ratio of the number of added virus particles to the number of cells is 1×10 5 , three wells were infected with each virus at the same MOI.

[0048] In addition, two control rAAVs were infected using the same infection method:

[0049] rAAV-C1: rAAV9-Ef1α-mCherry, the virus titer is 3.8×10 12 VG / mL;

[0050] rAAV-C2: rAAV9-TRE-mCherry-Ef1α-tTA, the virus titer is 5.8×10 12 VG / mL.

[0051] rAAV-C1 expresses mCherry without a peptide that promotes degradation, in contrast to rAAV-P1 and rAAV-P2; rAAV-C2 expresses mCherry driven by the TRE promoter, but introduces the TRE trans-acting factor tTA, which can enhance the promoter function of TRE, in contrast to rAAV-P3.

[0052] The infection and expression of the three experimental group viruses and the two control group viruses were observed and recorded 24 hours, 48 ​​hours, and 72 hours after infection.

[0053] The results of cell infection are shown in Figure 2. The number of cells with fluorescence expression caused by rAAV-P1 and rAAV-P2 infection was close to that of the control group rAAV-C1, but the fluorescence intensity was significantly reduced. Only a very small number of cells infected with rAAV-P3 were observed to express fluorescence, and the fluorescence intensity was significantly reduced compared with the control group rAAV-C2. For the three low-abundance fluorescence-expressing cells, their cell shape, size, and state were similar to those of the control group.

[0054] The above results show that the three adeno-associated viruses, rAAV-P1, rAAV-P2, and rAAV-P3, all successfully infected cells and expressed a small amount of fluorescent protein. Among them, rAAV-P1 and rAAV-P2 can use low-abundance fluorescence to label more cells, and rAAV-P3 can use low-abundance fluorescence to label sparse cells, and the three rAAVs have no negative impact on the cell state. In addition, cells expressing low-abundance fluorescent proteins are more clearly distinguishable from each other, and the location and status of individual cells can be better distinguished. Therefore, rAAV-P1, rAAV-P2, and rAAV-P3 can be used as tool viruses for the expression of low-abundance fluorescent proteins, and are particularly suitable for patch clamp experiments that require delicate manipulation of individual cells.

[0055] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for in vitro electrophysiological experiment, characterized in that: The steps of the method include: (1) Injecting rAAV expressing low-abundance fluorescent protein into target tissues or organs; The rAAV genome expressing the low-abundance fluorescent protein contains any one of the following a)-c): a) a promoter sequence, a fluorescent protein gene sequence and a PEST sequence; b) a promoter sequence, a fluorescent protein gene sequence and an ecDHFR DD sequence; c) a TRE promoter sequence and a fluorescent protein gene sequence, wherein the TRE promoter sequence is located upstream of the fluorescent protein gene sequence; (2) After the fluorescent protein has been expressed for a period of time, an in vitro electrophysiological experiment is performed on the target tissue or organ.

2. The method according to claim 1, characterized in that The in vitro electrophysiological experiment is a patch clamp experiment.

3. The method according to claim 1, characterized in that: a) the PEST sequence is located downstream of the fluorescent protein gene sequence and is located in the same expression frame as the fluorescent protein gene sequence, and the promoter sequence is located upstream of the fluorescent protein gene sequence and the PEST sequence; b) The ecDHFR DD sequence is located downstream of the fluorescent protein gene sequence and in the same expression frame as the fluorescent protein gene sequence, and the promoter sequence is located upstream of the fluorescent protein gene sequence and the ecDHFR DD sequence.

4. The method according to claim 1, characterized in that: The PEST sequence is shown as SEQ ID NO.1, the ecDHFR DD sequence is shown as SEQ ID NO.2, and the TRE promoter sequence is shown as SEQ ID NO.

1.

5. The method according to claim 1, characterized in that The rAAV genome expressing the low-abundance fluorescent protein also contains a transcriptional regulatory sequence.

6. The method according to claim 1, characterized in that The fluorescent protein is selected from any one of eGFP, BFP, CFP, GFP, YFP, RFP, iRFP, Cerulean, Venus, eGFP, eCFP, eYFP, eBFP, DsRed, dTomato, tdTomato, mCherry, mKate, mApple, mBanana, mCitrine, mOrange, mPlum, tagRFP, tagBFP, and HRP.

7. The method according to claim 1, characterized in that The promoter is selected from any one of CAG, CaMKIIα, CAR, CBA, CD68, c-fos, ChAT, CMV, CR, Ef1α, E-SARE, GAD67, GFAP, GFAP104, gfaABC1D, Grm6, hGRK1, hSyn, hUbC, LP1B, L7 / Pcp2, MBP, MCK, mDlX, mOXT, mTH, nEF, Nestin, NPY, Nrl, PGK, PV, RAM, RK, ROH, RPE65, SFRP2, SST, TBG, TCAP, TH, Thy1, TPH2, TRE, TRPV1, UAS, and Vgat.

8. The method according to claim 5, characterized in that The regulatory sequence is selected from any one of the transcriptional and post-transcriptional regulatory sequences WPRE, oPRE, cw3sl, SV40 polyA, hGH polyA, bGH polyA, and rbGlob polyA.

9. The method according to claim 1, characterized in that: The serotype of the rAAV expressing a low-abundance fluorescent protein is selected from any one of AAV1, AAV2, AAV2-Retro, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9-Retro, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV-PHP.eB, AAV-myo and their derived serotypes.

10. The use according to claim 1, characterized in that: The tissue is selected from the group consisting of nervous tissue, muscle, epithelial tissue, or connective tissue; Preferably, the neural tissue is selected from the central nervous system and its neural circuit-related tissues; The organ is selected from the group consisting of bladder, eye, ear, nose, oral cavity, tongue, pharynx, larynx, vomeronasal organ, salivary gland, liver, kidney, spleen, heart, intestine, stomach, pancreas, lung, trachea, blood vessel, lymphatic vessel, lymph node, limb, pituitary gland, thyroid gland, parathyroid gland, pancreatic islet, adrenal gland or reproductive organ.

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